1. Field of the Invention
This invention relates to novel methods for detecting and evaluating bladder cancer, utilizing hyaluronic acid (HA) and hyaluronidase (HAase).
2. Description of Related Art
Bladder carcinoma is the most common cancer of the urinary tract, accounting for 51,000 new cases and 11,000 deaths each year in the United States. Transitional cell carcinomas (TCCs) account for ≈90% of the bladder tumors. These tumors are heterogeneous in their ability to progress. For example, some TCCs behave in a benign fashion (low-grade, G1 tumors) whereas others are intermediate (G2 tumors) to highly aggressive (G3 tumors and carcinoma in situ (CIS)). The high-grade tumors generally metastasize quickly; indeed, at the time of clinical presentation (e.g., hematuria, irritative voiding symptoms etc.), invasive disease already exists for many patients with high-grade bladder tumors.
The two most important prognostic factors for TCCs are grade and stage (which indicates the depth of invasion) (American Joint Committee on Cancer: Staging of cancer at genito-urinary sites. In: Manual for Staging Cancer, 3rd edition, pp. 194-195, J. B. Lippincott Co., Philadelphia, 1988). Low-grade (G1) tumors are mostly confined to the mucosa (stage Ta) and have a  less than 2% chance of progression (Heney, Natural history of superficial bladder cancer. Urol. Clin. North Am., 19: 429-435, 1992; Heney and Flanagan, Superficial bladder cancer progression and recurrence. J. Urol., 130: 1083-1086, 1983). Intermediate-grade (G2) tumors range from being non-invasive (Ta) to invasive (stages T1-T4). The G2, Ta tumors have xcx9c11% chance of progression (Heney, Natural history of superficial bladder cancer. Urol. Clin. North Am., 19: 429-435, 1992). With the exception of carcinoma in situ (CIS), most high-grade tumors are initially detected at least at stage T1 (invading lamina propria) and are thus invasive. Muscle invasion (stage T2) by the tumor is ominous, as 50% of these patients develop distant metastasis within two years of diagnosis despite radical surgery, and 60% of them die within 5 years, however treated (Heney and Flanagan, Superficial bladder cancer progression and recurrence. J. Urol., 130: 1083-1086, 1983; Friedell et al., Summary of workshop on carcinoma-in-situ of the bladder. J. Urol., 136: 1047-1048, 1986; Soloway, Invasive bladder cancer: Selection of primary treatment. Semin. Oncol., 17: 551-554, 1990). Due to the malignant nature of high-grade TCCs, their early detection prior to muscle invasion, is crucial for a favorable prognosis.
Thus, it is not only important to detect the presence of tumor early, it is also crucial to identify the high-grade tumors which present with such a grim prognosis. Tumor recurrence is also a characteristic of bladder carcinoma. Therefore, despite a complete remission of the original tumor, patients must be closely followed in order to monitor the treatment efficacy and recurrence (Heney, Natural history of bladder cancer. Urol. Clin. North Am., 19: 429-433, 1992).
The current methods for bladder cancer detection involve cystoscopy, bladder washings, and biopsy. These procedures are invasive and require some form of anesthesia. Urine cytology use is possible but its specificity is low due to its subjective nature. A few other markers such as DNA ploidy, p53 mutations, microsatellite DNA, xcex2-glucuronidase, basic-FGF levels, autocrine motility factor receptor etc. have been shown to be associated with bladder cancer (Sidransky and Messing, Molecular genetics and biochemical mechanisms in bladder cancer. Urol. Clin. North Am., 19: 629-639, 1992; Mao et al., Molecular detection of primary bladder cancer by microsatellite DNA. Science, 271: 659-662, 1996; Nguyen et al., Elevated levels of the angiogenic peptide basic fibroblast growth factor in urine of bladder cancer patients. J. Natl. Cancer Inst., 85: 241-242, 1993; Esrig et al., Accumulation of nuclear p53 and tumor progression in bladder cancer. N. Engl. J. Med., 331: 1259-1264, 1994; Ho, Urinary xcex2-glucuronidase in screening and follow up of primary urinary tract malignancy. J. Urol., 154: 1335-1338, 1995; Korman et al., Autocrine motility factor receptor as a possible urine marker for transitional cell carcinoma of the bladder. J. Urol., 154: 347-349, 1995). However, most of these have not yet been used clinically as diagnostic markers.
Currently, three non-invasive diagnostic tests for bladder cancer are under investigation in the United States. The hematuria home screening test has high sensitivity but low specificity due to the wide spectrum of benign genito-urinary (GU) conditions (kidney stones, benign prostatic hyperplasia etc) which give rise to false positives (Britton et al., A community study of bladder cancer screening by the detection of occult urinary bleeding. J. Urol., 148:788-790, 1992; Messing et al., Hematuria home screening: Repeat testing results. J. Urol., 154: 57-61, 1995). The second test is the Bard BTA Latex agglutination assay. A multi-center trial for this test was conducted in the United States to monitor bladder tumor recurrence. The results show that the sensitivity of this test to detect bladder tumors and, more importantly high-grade TCCs, is only ≈40-50% (Sarosdy et al., Results of a multicenter trial using the BTA test to monitor for and diagnose recurrent bladder cancer, J. Urol., 154: 379-384, 1995; U.S. Pat. No. 5,264,370). In another multicenter study involving 90 patients and the third test, Soloway et al. have shown that the NMP22 test has an overall sensitivity of 70% to detect bladder tumor recurrence (Soloway et al., Use of a new tumor marker NMP22 in the detection of occult or rapidly recurring transitional cell carcinoma of the urinary tract following surgical treatment. J. Urol., 156: 363-367, 1996). Thus, to date, no non-invasive test exists that can detect bladder tumor and/or evaluate its grade with high sensitivity and specificity.
With the above problems in the art in mind, the inventors have developed non-invasive methods to detect bladder cancer by measuring the levels of certain xe2x80x9cmolecular determinantsxe2x80x9d specifically expressed in the biological fluids (such as urine specimens) of bladder cancer patients. More particularly, the methods of the invention are based on the inventors"" discovery that levels of hyaluronic acid and hyaluronidase in a sample of biological fluid, especially urine, are associated with the presence and grade of bladder cancer.
Hyaluronic acid (also known in the art as hyaluronate and hyaluronan, and abbreviated as HA), is a glycosaminoglycan comprising a straight unbranched polysaccharide chain with alternating units of N-acetyl-D-glucosamine and D-glucuronic acid. HA is present ubiquitously in various types of biological material, including both bacteria and animals. In humans, HA is found in high concentrations in umbilical cords, vitreous humor of the eyes, cartilage and synovial fluid. Small amounts of HA are present in CSF, lymph, blood, serum and urine. Levels of HA have been associated with diseases such as rheumatoid arthritis, liver cirrhosis, and Wilms"" tumor. HA is associated with non-specific tumors in general, but its use has not been applied heretofore to the discovery, therapy and management of particular clinical tumors.
HA has been known to play a role in several pathophysiological conditions including cancer. For example, HA levels have been shown to be elevated in certain animal tumor models (e.g., rabbit V2 carcinoma, Knudson et al., The role and regulation of tumor associated hyaluronan. In: The Biology of Hyaluronan (J. Whelan, ed.), pp. 150-169, New York, Wiley Chichister (Ciba Foundation Symposium 143), 1989) and human cancers (e.g., lung, Wilms"" tumor, breast, etc., Knudson et al., ibid.).
In tumor tissues, HA expands upon hydration opening spaces for tumor cell migration (Knudson et al., The role and regulation of tumor associated hyaluronan. In: The Biology of Hyaluronan (J. Whelan, ed.), pp. 150-169, New York, Wiley Chichister (Ciba Foundation Symposium 143), 1989). Furthermore, tumor cells migrate on HA matrix by interacting through certain cell surface receptors (e.g., CD44; Thomas et al., Migration of human melanoma cells on hyaluronate is related to CD44 expression. J. Invest. Dermatol., 100: 115-120, 1993). HA also forms a halo around tumor cells that protects them against immune surveillance (Hobarth et al., Topical chemoprophylaxis of superficial bladder cancer by mitomycin C and adjuvant hyaluronidase. Europ. Urol., 21: 206-210, 1992). More recently, small fragments of HA (xcx9c3-25 disaccharide units) have been shown to promote angiogenesis (West et al., Angiogenesis induced by degradation products of hyaluronic acid. Science, 228: 1324-1326, 1985; West and Kumar, The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity. Exp. Cell Res., 183: 179-196, 1989). Earlier work from one of the inventors has shown that an HA fragment of 10-15 disaccharide units (F1 fragment), stimulates proliferation of bovine aortic endothelial cells. A similar sized fragment has also been shown to promote endothelial cell migration and tubule formation (Banarjee and Toole, Hyaluronan binding protein in endothelial cell morphogenesis. J. Cell Biol., 119: 643-652, 1992).
In the present application, the inventors measure the HA levels in the urine of normal individuals and bladder cancer patients, and in the extracts prepared from normal bladder and tumor tissues, as discussed below. Also as discussed below, the inventors examine the profile of HA species present in the urine of normal individuals, bladder cancer patients, and patients with other genito-urinary (GU) conditions. In addition, they determine whether the HA or HA fragments present in the urine affect the proliferation of human endothelial cells.
The inventors disclose in the present application that the urinary HA levels of bladder cancer patients with G1, G2 and G3 tumors are significantly elevated (for instance, 4-9 fold elevation) as compared to those of normal individuals and patients with other genito-urinary (GU) conditions (P less than 0.001). As discussed below, the inventors also discovered that a comparison of pre- and post-treatment urinary HA levels can be used to monitor treatment efficacy. For example, elevated post-treatment urinary HA levels are indicative of persistent bladder cancer and possible relapse at later time. The urinary HA levels are also useful to monitor bladder cancer recurrence during follow-up visits subsequent to the initial treatment. The increase in urinary HA concentration is a direct correlate of the elevated tumor-associated HA levels, because the HA levels are also elevated (for instance, 3-5 fold) in bladder tumor tissues (P less than 0.001). The profiles of urinary HA species of normal individuals and bladder cancer patients are different. While only the intermediate size HA species are found in the urine of normal and low-grade bladder tumor patients, the urine of high-grade bladder cancer patients contains both the high molecular mass and the small angiogenic HA fragments.
The inventors also disclose in the present application that these urinary HA fragments stimulate a mitogenic response (for instance, 2-4 fold) in primary human microvessel endothelial cells, suggesting that the small HA fragments may regulate tumor angiogenesis by modulating endothelial cell functions. The inventors hypothesized that small HA fragments in the urine of high-grade bladder cancer patients may indicate that a hyaluronidase activity is present in the urine of these patients.
Hyaluronidase (HAase) is an endoglycosidic enzyme that degrades HA by hydrolyzing the N-acetylglucosaminic bonds in HA. The limited degradation of HA by hyaluronidase results in the generation of HA fragments of specific lengths (xcx9c3-25 disaccharide units) that are angiogenic (West et al., Angiogenesis induced by degradation products of hyaluronic acid. Science, 228: 1324-1326, 1985). In vertebrates, hyaluronidases can be categorized into two classes, those active at neutral pH (pH optimum 5.0), and those active at acidic pH (pH 3.5-4.0) (Roden et al., Enzymatic pathways of hyaluronan catabolism. In: The Biology of hyaluronan, (J. Wh.elan, ed.), pp. 60-86, New York, Wiley Chichister (Ciba Foundation Symposium 143), 1989; West et al., ibid.; Gold, Purification and properties of hyaluronidase from human liver. Biochem. J., 205: 69-74, 1982; Fraser and Laurent, Turnover and metabolism of Hyaluronan. in: Biology of Hyaluronan, (J. Whelan, ed.), pp. 41-59, New York, Wiley Chichister (Ciba Foundation Symposium 143), 1989; Zhu et al., Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum heme-binding protein. J. Biol. Chem., 269: 32092-32097, 1994; Lin et al., A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus layer surrounding the egg. J. Cell Biol., 125: 1157-1163, 1995). For example, the testicular hyaluronidase is of neutral type whereas the liver hyaluronidase has an acidic pH optimum. The concerted actions of both HA and hyaluronidases are known to play important roles during embryonic development, vasculogenesis, vascular remodeling, immune surveillance and tumor progression (McCormick and Zetter, Adhesive interactions in angiogenesis and metastasis. Pharmacol. Ther., 53: 239-260, 1992; Hobarth et al., Topical chemo-prophylaxis of superficial bladder cancer by mitomycin C and adjuvant hyaluronidase, Eur. Urol., 21: 206-210, 1992; Knudson et al., The role and regulation of tumor-associated hyaluronan. In: The Biology of Hyaluronan (J. Whelan, ed.) pp. 150-169, New York, Wiley, Chichester (Ciba Foundation Symposium 143), 1989; Lin et al., Urinary hyaluronic acid is a Wilms"" tumor marker. J. Ped. Surg., 30: 304-308, 1995; Stern et al., Hyaluronidase levels in urine from Wilms"" tumor patients. J. Natl. Canc. Inst., 83: 1569-1574, 1991). The inventors have shown that hyaluronidase levels are elevated in prostate cancer and the increase correlates with the aggressiveness of prostate cancer (Lokeshwar et al., Association of hyaluronidase, a matrix-degrading enzyme with prostate cancer progression. Cancer Res., 56: 651-657, 1996).
The inventors disclose in the present application that the urinary HAase levels of bladder cancer patients with G2 and G3 tumors are significantly elevated (for instance, 5-8 fold) as compared to those of normal individuals, patients with G1 tumors and patients with other GU conditions (P less than 0.001). As discussed below, the inventors also discovered that a comparison of pre- and post-treatment urinary hyaluronidase levels can be used to monitor treatment efficacy. For example, post-treatment elevated urinary hyaluronidase levels are indicative of persistent G2 or G3 bladder tumor and possible relapse at a later time. The urinary hyaluronidase levels are also useful to monitor G2 or G3 bladder tumor recurrence during follow-up visits subsequent to the initial treatment. The increase in urinary hyaluronidase levels is due to the secretion of a tumor-associated hyaluronidase into the urine, as the hyaluronidase levels in G2/G3 tumor tissues are also higher (for instance, about 6-7 fold) than those in normal bladder and G1 tumor tissues (P less than 0.001). The bladder tumor-associated hyaluronidase activity is distinct from other hyaluronidases, has a pH optimum of 4.3 and is attributed to two proteins of Mr 65 kD (p65) and 55 kD (p55).
Prior to the invention, neither HA nor HAase have been associated with bladder cancer, nor have they been used for the detection or evaluation of bladder cancer. However, using the assay methods of the present invention, HA and HAase can be used in a non-invasive test to detect bladder cancer and evaluate its particular grade.
ELISA-like assays to determine HA concentrations have been described previously. For example, Goldberg et al. (U.S. Pat. No. 5,378,637) have described that HA can be measured in a biological sample by coating a solid support with HA, incubating the sample with a cartilage proteoglycan (which is known to bind HA in any biological material), and then exposing the sample to a coated solid support. The amount of cartilage proteoglycan bound to the solid HA support is determined by anti-keratin sulfate-reactive antibody.
A similar method has been described using pig laryngeal cartilage proteoglycan by Fosang et al. (Matrix, 10: 306-313, 1990). That article describes an ELISA plate-based assay for hyaluronan using biotinylated proteoglycan G1 domain (HA-binding region).
An ELISA-like assay has been described by Stern and Stern (Matrix, 12: 397-403, 1992). That reference describes hyaluronidase determination using biotinylated HA binding protein, mouse anti-keratin sulfate antibody, biotinylated goat anti-mouse IgG and avidin-biotin detection system. Using the same assay, Stern et al. have measured urinary hyaluronidase levels in Wilms"" tumor patients (Stern et al., Hyaluronidase levels in urine from Wilms"" tumor patients. J. Natl. Canc. Inst., 83: 1569-1574, 1991).
Other hyaluronic acid determination methods are described by Chichibu (U.S. Pat. No. 5,019,498), and Brandt et al. (U.S. Pat. No. 4,826,776).
The methods of the present invention are based on the discovery that urinary HA and HAase levels are diagnostic markers for the detection of bladder cancer, evaluation of its grade and monitoring of the efficacy of its treatment.
With the invention, the measurements of HA and HAase levels are technically simple, because these are ELISA-like assays. Both assays require only an HA-binding protein, which can be purified in large quantities using a well established procedure (Tengblad, A. Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage. Biochim. Biophys. Acta, 578: 281-289, 1979). Both assays are simple, non-invasive yet highly sensitive and specific tests that may be used clinically for bladder cancer detection. The ELISA-like assay for HA measurement (HA test) detects bladder cancer regardless of the tumor grade. The ELISA-like assay for HAase measurement (HAase test) preferentially detects intermediate-grade (G2) to high-grade (G3) bladder tumors.
Since urinary hyaluronidase measurement detects CIS (pre-invasive G3 bladder tumors) as well as G2, Ta tumors, the invention is a better non-invasive method for the early detection of G2 and G3 bladder tumors which present with poor prognosis for the patient.
Accordingly, in one embodiment of this invention bladder cancer is tested for by quantitatively measuring HA in a sample of biological fluid (such as, for instance, a urine specimen) collected from a patient suspected of having bladder cancer. Any conventional assay methodology can be used to determine the presence and measurement of HA, including radioassays, sandwich assays, inhibition assays and the like. However, HA is preferably measured a competitive binding assay. More preferably, the assay of the invention works in the same manner as an ELISA test, but does not make use of antibody completing mechanisms.
For instance, bladder cancer can be detected in an assay method comprising the steps of:
(a) coating a solid support (preferably, microtiter wells) with HA;
(b) contacting and incubating HA binding protein (HABP) with the coated solid support in the presence of a sample of biological fluid (such as a urine sample) collected from a person suspected of having bladder cancer, under conditions such that the HABP is permitted to bind to the HA coated on the solid support and the HA in the sample (if any is present);
(c) determining the amount of HABP bound to the HA coated on the solid support, and determining therefrom the amount of HA present in the sample.
In this embodiment, the coated HA and the HA contained in the sample xe2x80x9ccompetexe2x80x9d to bind with the HABP. Where HA is present in the sample, less HABP will bind to the coated HA, as determined by, for instance, comparison with a standard. In other words, little HABP bound to the coated HA would mean HA present in the sample, which would be indicative of bladder cancer.
The preferred way to determine the amount of HABP bound to the HA coated on the solid support, and determine therefrom the amount of HA present in the sample, is to detect a signal associated with or produced by the bound HABP.
For example, a microtiter plate reader can be used to measure absorbance of colored product as an indirect measure of biotinylated HABP bound to the solid support (avidin-enzyme conjugate and labeled substrate are used to generate the colored product). The maximum absorbance can be obtained by incubating the HA-coated wells with buffer alone in the absence of any HA or HA-containing sample. A standard graph can then be prepared by plotting absorbance versus ng/well or 0.2 ml of HA. Using this standard graph, the HA concentration (ng/ml) in each dilution of the sample can be calculated. From several such determinations the mean HA concentration in each sample can be determined. Protein concentration (mg/ml) of the sample can be determined, for example, by automated analysis or with a protein assay kit (BioRad, Richmond, Calif.). The HA concentrations can be normalized to the protein content and expressed as ng/mg protein.
For example, the calculations for determining urinary HA levels can be as follows: Axc3x97dilution factor÷mg/ml urinary protein, where A is ng/ml of HA concentration extrapolated from the standard graph. The HA levels are finally expressed as ng/mg total protein. A low absorbance reading would be indicative of a significant amount of HA in the urine sample, which would itself be indicative of bladder cancer in the patient.
In general, a calculation of more than about 500 ng/mg HA in the sample is indicative of bladder cancer in the patient.
The sensitivity of this method to detect bladder cancer using HA can be about 88% or more (and may be as high as 100%) and the specificity can be about 87% or more (and may be as high as 100%). For this invention, specificity is understood to be a measurement of false positives, where a specificity of 100% means there are no false positives (i.e., no suggestion of the presence of bladder cancer when the patient does not in fact have bladder cancer). Sensitivity is understood to be a measurement of false negatives, where a sensitivity of 100% means there are no false negatives (i.e., no suggestion that there is no bladder cancer when the patient in fact does have bladder cancer).
In another embodiment of this invention, bladder cancer is tested for and its grade evaluated by quantitatively measuring HAase in a sample of biological fluid (such as a urine specimen) collected from a patient suspected of having bladder cancer. As with HA, any conventional assay methodology can be used to determine the presence and measurement of HAase, including radioassays, sandwich assays, inhibition assays and the like. However, HAase is preferably measured by a competitive binding assay. More preferably, the assay of the invention works in the same manner as an ELISA test, but does not make use of antibody completing mechanisms.
For instance, bladder cancer can be detected in an assay method comprising the steps of:
(a) coating a solid support (preferably, microtiter wells) with HA;
(b) contacting and incubating a biological sample collected from a person suspected of having bladder cancer with the coated solid support, under conditions such that the HAase present in the sample (if any is present) is permitted to degrade the HA coated on the solid support;
(c) contacting and incubating HA binding protein (HABP) with the coated solid support, under conditions such that the HABP is permitted to bind to any non-degraded HA coated on the solid support;
(d) determining the amount of HABP bound to the HA coated on the solid support, and determining therefrom the amount of HA present in the sample.
In this embodiment, where HAase is present in the sample, it will degrade the coated HA and permit less HABP to bind to the coated HA, as determined by, for instance, comparison with a standard. A low measurement of HABP bound to the coated HA, then, would be indicative of either intermediate or high grade bladder cancer.
The preferred way to determine the amount of HABP bound to the HA coated on the solid support, and determine therefrom the amount of HAase present in the sample, is to detect a signal associated with or produced by the bound HABP.
For example, a microtiter plate reader can be used to measure absorbance of colored product as an indirect measure of biotinylated HABP bound to the solid support (avidin-enzyme conjugate and labeled substrate are used to generate the colored product). The maximum absorbance can be obtained by incubating the HA-coated wells with buffer alone in the absence of any HAase or HAase-containing sample. A standard graph can be prepared by plotting absorbance versus mU/ml of Streptomyces HAase. Using this standard graph, the HAase concentration (mU/ml) in each dilution of the sample can be calculated. From several such determinations the mean HAase concentration in each sample can be determined. Protein concentration (mg/ml) of the sample can be determined by automated analysis or with a protein assay kit (BioRad, Richmond, Calif.). The HAase concentrations can be normalized to the protein content and expressed as mU/mg protein.
The calculations for determining urinary HAase levels can be as follows: Axc3x97dilution factor÷mg/ml urinary protein, where A is mU/ml of HAase concentration as extrapolated from the standard graph. The HAase levels are finally expressed as mU/mg total protein. A low absorbance reading would indicate a high amount of HAase present in the urine sample, which itself would indicate intermediate- or high-grade bladder cancer in the patient.
In general, a calculation of more than about 10 mU/mg HAase is indicative of intermediate or high grade bladder cancer in the patient.
The sensitivity of this method to detect the intermediate- to high-grade bladder cancer can be about 85% or more (and may be as high as 100%) and the specificity can be about 88% or more (and may be as high as 100%).
To detect low-grade bladder cancer, a combination of HA and HAase tests may be used. A calculation of less than about 10 mU/mg HAase (corresponding to a higher amount of HABP bound to the coated HA) is indicative of either low grade bladder cancer or no bladder cancer at all. This embodiment of the invention utilizing an HAase assay does not distinguish between the presence of low grade bladder cancer or no bladder cancer at all. Consequently, it is preferred that the HA assay be used in conjunction with the HAase assay to test a patient, because the HA assay can detect the presence of even low grade bladder tumors, although it does not distinguish between particular grades. Thus, in order to detect and diagnose a low grade bladder tumor, both the HA and the HAase assays should be run. The HA assay would give a positive result (i.e., levels of HA exceeding about 500 ng/mg, indicating the presence of a tumor), while the HAase assay would give a negative result (i.e., levels of HAase less than about 10 mU/mg, indicating that there is no intermediate or high grade tumor present).
In a further embodiment, the invention relates to diagnostic kits for testing and evaluating bladder cancer. The kit comprises HA and/or HAase, HABP and a marker or HABP conjugated to a marker, and ancillary reagents suitable for use in detecting the presence of HA and/or HAase in a biological sample. An example of a diagnostic kit contemplated by this invention is a conventional dipstick test device.
These and other embodiments of the instant invention are described in further detail below.